Heparan sulfate is a highly sulfated polysaccharide with very complicated saccharide structures. Heparan sulfate is present on the cell surface and in the extracellular matrix in a large quantity, and plays important roles in a wide range of physiological and pathophysiological functions. We propose to understand the contribution of specific sulfonated saccharide sequences to assist herpes simplex virus infections. The binding of heparan sulfate to the herpes simplex virus envelope proteins represents the initial step of viral infections. Recent studies reveal that a specific 3-O-sulfated heparan sulfate plays an essential role for assisting the entry of herpes simplex virus 1. This unique subtype of heparan sulfate is biosynthesized by several heparan sulfate 3-0-sulfotransferase isoforms (3-OST). We plan to investigate the susbtrate recognition mechanism of 3-OST. We will also investigate how to prepare the polysaccharides or oligosaccharides with unique sulfonation patterns to improve the efficacy and slectivity for inhibiting herpes simplex infections. Three specific aims are proposed in this project: Specific Aim 1 is to investigate the substrate recognition mechanism of 3-OST. We plan to conduct a crystal structure study on 3-OST-5 and site-directed mutagenesis. Second, we plan to convert 3-OST-1, an enzyme that does not produce an HSV entry receptor, to a 3-OST-3-like enzyme that generates an HSV entry receptor by random mutagenesis. Specific Aim 2 is to develop an enzymatic approach to synthesize octasaccharides and polysaccharides that carry the critical 3-0-sulfo glucosamine residues. Our recent data suggest that this approach allows us to synthesize the polysaccharides in sufficient amounts for studying their effects on inhibiting herpes simplex virus infections. Specific Aim 3 is to determine the efficacy of the synthesized compounds on inhibitng herpes simplex infections. We plan to determine their effects on the entry as well as on the binding of herpes simplex virus to the target cells. We also plan to investigate the efficacy of these compounds on the entry of herpes simplex virus via different cellular receptors. Our results could lead to a novel approach to treat herpes simplex virus infections and improve the basic understanding on the biosynthesis of HS with specific sulfonation patterns.